Rotaviruses are important causes of gastrointestinal disorders and diarrhea in avian and animal species, including man. There are at least eleven known serotypes of rotavirus, some of which are found in humans and others found in various animals and birds. There appears to be some cross-protection among strains, but this is not complete. A number of the important capsid proteins have been sequenced and show considerable homology among serotypes.
The rotavirus genome is thought to consist of eleven segments of double-stranded RNA regardless of the strain. A designation system for the proteins encoded by these segments and present in the intact virus has been proposed, but has not remained constant over the years. There appear to be three proteins associated with the inner shell (VP1, VP2, and VP6) and three associated with the outer capsid. Of interest herein are the inner capsid protein VP6 and two of the outer capsid proteins, VP7 and VP4 (designated VP3 in former nomenclature).
(In various rotaviruses, the absolute order of the genomic fragments does not always conform to the same genes; for example in rotavirus strains SA11, W, and Wa, gene 9 codes for VP7, while in rotavirus strains DS-1, and UK bovine rotavirus (BRV) gene 8 codes for VP7. Also, especially for bovine and human rotavirus, there are variations in the mobility of proteins derived from different isolates originating from the same species. However, the identity of the various virus proteins (VP) designated by the foregoing designations is not in doubt for any particular species.)
As the invention concerns vaccines comprised of epitopes from VP7, VP4 and VP6, further descriptions of these particular proteins are provided as follows:
The major glycoprotein of the outer shell, VP7, has an approximate apparent molecular weight of 38.2 kd in its unreduced form and 41.9 kd in its reduced form and has been shown to be the major antigen responsible for inducing neutralizing antibodies to the virus, and for attachment to cells. A 14 kd fragment of this protein from BRV was also shown to raise neutralizing antibodies against the virus. (Sabara, M., et al., J Virol (1985) 53:58-66). Regions of conservation among strains infecting human and animal species in this protein have been reviewed by Estes, M. K., et al., Microbiol Rev (1989) 53:410-449. The complete amino acid sequence of rotavirus VP7 glycoprotein for three strains of rotavirus, and a partial sequence for a fourth strain are shown in FIG. 1.
Gunn, P. R., et al., J Virol (1985) 54:791-797, investigated various putative antigenic determinants in VP7 of the SA11 serotype. These authors compared the amino acid sequences of the VP7 protein of SA11, NCDV, S2 and Wa serotypes. They investigated the immunogenicity of peptides corresponding to positions 66-76; 90-103; 174-183; 208-225; 247-259; and 275-295 conjugated to keyhole limpet hemacyanin (KLH) using 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide (EDCI) with mixed results. All of the peptides were capable of generating antisera immunoreactive with the immunizing peptides themselves and the denatured form of VP7. However, high titer polyclonal neutralizing antiserum directed against the whole SA11 virus did not recognize any of the seven peptides when they were immobilized on microtiter dishes, pretreated with glutaraldehyde. Further, in a virus neutralization assay, where control polyclonal antiserum prepared against whole virus had a neutralization titer of 10.sup.6 U/ml, none of the peptide antisera showed significant neutralizing activity. Further, none of the sera recognized whole virus in solid phase RIA. The authors concluded that in the native virus the regions represented by these peptides were not properly exposed, and suggested the possibility of the antigenic sites being composed of discontinuous determinants.
The various serotypes of rotavirus are defined by the neutralizing activity stimulated by VP7; on this basis, eleven serotypes have been identified, six of which are found in humans.
VP4 (formerly called VP3) is also an outer capsid protein and has an approximate apparent molecular weight of 82 kd in unreduced form and 84 kd in reduced form. FIG. 3 shows the amino acid sequences of two strains of rotavirus--C486 which is a bovine strain and SA11 which is a simian-infecting strain.
The inner capsid protein of interest herein is VP6 which is a 45 kd molecular weight protein. Antibodies raised against VP6 appear to be the most cross-species reactive among those raised to viral proteins. The nucleotide sequence and deduced amino acid sequence of the SA11 VP6 protein, as disclosed by Estes et al., Nucleic Acids Res (1984) 12:1875-1887 is shown in FIG. 3. In addition to being itself immunogenic, VP6 has been shown, at least partially by virtue of its abilities to form aggregates of various shapes, to be a superior carrier for other haptens. This is disclosed EPO application 87/3077465.
The present invention provides subunit peptide vaccines corresponding to the epitopic regions of VP7, VP4, and VP6 which are effective as vaccines in that they show demonstrated protection against challenge and/or elicit the production of neutralizing antibodies. The use of these subunit vaccines confers a number of advantages, including safety, economy, and effectiveness.